Characteristics of water and ion exchange of Elodea nuttallii cells at high concentrations of lanthanides.

Changes of diffusive permeability of membranes of Elodea nuttallii cells following a short-term (60 min) treatment with high concentrations of lanthanides were recorded by the 1H NMR-diffusometry and conductometry methods. The 1-h infiltration of segments of Elodea nuttallii internodes in 10 mM solutions of nitrates of La, Nd and Lu resulted in the increased leakage of electrolytes from cells, but has no effect on a water diffusive permeability of membranes. In samples subjected to a 30 min pretreatment with a water channel inhibitor HgCl2 the water diffusive permeability of membranes (Pd) drops down under the influence of lanthanides, as well as an outcome of electrolytes. To explain the observed effects the change of spontaneous curvature of membrane lipid layer has been taken into consideration. The interaction of lanthanides with lipids of plasmalemma leads to the negative spontaneous curvature of lipid layer at which membrane channels are unclosed. Blocking of the ionic and water channels by mercury ions compensate the effect of change of spontaneous curvature of lipid layer.

Kinetics of nickel bioaccumulation and its relevance to selected cellular processes in leaves of Elodea canadensis during short-term exposure.

Elodea canadensis is an aquatic macrophyte used widely as a bioindicator for the monitoring of water quality and in the phytoremediation of metal-contaminated waters. This study considers the kinetics of nickel bioaccumulation and changes in accompanying metabolic and stress-related physiological parameters. These include photosynthetic activity, pigment content, the accumulation of thiol-containing compounds, thiobarbituric acid-reactive substance (TBARS) products, and the activity of selected antioxidant enzymes (catalase, glutathione reductase, superoxide dismutase). Elodea leaves accumulated nickel according to pseudo-second-order kinetics, and the protective responses followed a time sequence which was related to the apparent rates of nickel accumulation. The applicability of second-order kinetics to the Ni uptake by Elodea leaves during the first 8 h of exposure to the metal suggested that the passive binding of metal ions (chemisorption) was a rate-limiting step at the initial phase of Ni accumulation. This phase was accompanied by an increase in photosynthetic activity together with elevated photosynthetic pigments and protein synthesis, the enhanced activity of antioxidant enzymes, and increased thiol concentration. In contrast, there was a decrease in metabolic activity upon the accumulation of TBARS, and the decline in enzyme activity was observed in the saturation phase of Ni accumulation (8-24 h). These results show that a correlation exists between the protective response and the apparent kinetic rate of Ni uptake. Thus, the time of exposure to the toxicant is a crucial factor in the activation of specific mechanisms of Ni detoxification and stress alleviation.

A lysigenic programmed cell death-dependent process shapes schizogenously formed aerenchyma in the stems of the waterweed Egeria densa.

BACKGROUND AND AIMS: Plant adaptation to submergence can include the formation of prominent aerenchyma to facilitate gas exchange. The aim of this study was to characterize the differentiation of the constitutive aerenchyma in the stem of the aquatic macrophyte Egeria densa (Hydrocharitaceae) and to verify if any form of cell death might be involved. METHODS: Plants were collected from a pool in a botanical garden. Aerenchyma differentiation and apoptotic hallmarks were investigated by light microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay coupled with genomic DNA extraction and gel electrophoresis (DNA laddering assay). Cell viability and the occurrence of peroxides and nitric oxide (NO) were determined histochemically using specific fluorogenic probes. KEY RESULTS: Aerenchyma differentiation started from a hexagonally packed pre-aerenchymatic tissue and, following a basipetal and centripetal developmental pattern, produced a honeycomb arrangement. After an early schizogenous differentiation process, a late lysigenous programmed cell death- (PCD) dependent mechanism occurred. This was characterized by a number of typical apoptotic hallmarks, including DNA fragmentation, chromatin condensation, apoptotic-like bodies, partial cell wall lysis and plasmolysis. In addition, local increases in H2O2 and NO were observed and quantified. CONCLUSIONS: The differentiation of cortical aerenchyma in the stem of E. densa is a complex process, consisting of a combination of an early schizogenous differentiation mechanism and a late lysigenous PCD-dependent process. The PCD remodels the architecture of the gas spaces previously formed schizogenously, and also results in a reduction of O2-consuming cells and in recycling of material derived from the lysigenic dismantling of the cells.

Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells.

In the outer periclinal cytoplasm of leaf epidermal cells of an aquatic angiosperm Vallisneria, blue light induces "chloroplast de-anchoring", a rapid decline in the resistance of chloroplasts against centrifugal force. Chloroplast de-anchoring is known induced within 1 min of irradiation with high-fluence-rate blue light specifically, preceding the commencement of chloroplasts migration toward the anticlinal cytoplasm. However, its regulatory mechanism has remained elusive, although pharmacological analysis suggested that a calcium release from intracellular calcium stores is necessary for the response. In search of the responsible photoreceptors, immunoblotting analysis using antibodies against phototropins demonstrated that cross-reactive polypeptides of 120-kDa exist in the plasma-membrane fraction prepared from the leaves. In vitro phosphorylation analysis revealed that 120-kDa polypeptides were phosphorylated by exposure to blue light in a fluence-dependent manner. The blue-light-induced phosphorylation activity was sensitive to a Ser/Thr kinase inhibitor, staurosporine, and unusually was retained at a high level for a long time in darkness. Furthermore, phototropin gene homologs (Vallisneria PHOTOTROPIN1 and PHOTOTROPIN2) expressed in leaves were isolated. We propose that calcium-regulated chloroplast de-anchoring, possibly mediated by phototropins, is an initial process of the blue-light-induced avoidance response of chloroplasts in Vallisneria.

Cellular proton dynamics in Elodea canadensis leaves induced by cadmium.

Our earlier investigations showed that Elodea canadensis shoots, grown in the presence of cadmium (Cd), caused basification of the surrounding medium. The present study was aimed to examine the proton dynamics of the apoplastic, cytosolic and vacuolar regions of E. canadensis leaves upon Cd exposure and to establish possible linkage between cellular pH changes and the medium basification. The changes in cytosolic calcium [Ca(2+)]cyt was also investigated as the [Ca(2+)]cyt and [pH]cyt homeostasis are closely linked. The cellular H(+) and Ca(2+) concentrations were monitored by fluorescence microscopy and ion-specific fluorescent dyes. Cadmium concentration of leaf-cell walls was measured after plant cultivation at different fixed levels of starting pH. The protoplasts from E. canadensis leaves were isolated by use of a newly developed enzymatic method. Upon Cd addition, both cytosolic and vacuolar pH of leaf protoplasts increased with a concomitant rise in the cytosolic Ca(2+) concentration. Time course studies revealed that changes in [Ca(2+)]cyt and [pH]cyt followed similar dynamics. Cadmium (0.5 µM) exposure decreased the apoplastic pH by 0.85 units. The maximum cell wall bound Cd-contents were obtained in plants grown at low starting pH. It is concluded that Cd treatment causes apoplastic acidosis in E. canadensis leaves associated with enhanced Cd binding to the cell walls and, consequently, reduced Cd influx into the cytosol.

Laser desorption/ionization mediated by bionanostructures from microalgae.

RATIONALE: Organic matrices are the state-of-the-art ionization mediators in Laser Desorption/Ionization Mass Spectrometry (LDI-MS). Despite improvements in understanding matrix chemistry, interfering matrix-related signals complicate the analysis. Surface-assisted LDI techniques like desorption/ionization on silicon (DIOS) or nanostructure initiator mass spectrometry (NIMS) provide promising alternatives but rely often on elaborate materials. METHODS: We introduce nanopatterned biomineralized cell walls of microalgae as easily accessible biological surfaces that support the ionization of embedded molecules in LDI-MS. Microalgae cell walls were cleaned through oxidation and washing before pipetting on a stainless-steel matrix-assisted laser desorption/ionization (MALDI) target. Added molecules were efficiently ionized in positive and negative ionization mode in common MALDI sources. The method was rigorously validated by comparison with established MALDI experiments. RESULTS: Ionization of PEG600, D-sphingosine and raffinose was successfully mediated by nanostructured cell wall preparations from two different microalgae. Without any change in protocol, steric acid could be detected in the negative ionization mode. Ionization is also supported by commercially available celite, a material containing mineralized diatom cell walls. Characteristic ingredients of fresh coffee were detected in LDI-MS after pipetting it on celite without further sample preparation. Caffeine and saccharose were detected in positive and characteristic fatty acids in negative ionization mode. Detection limits were comparable to established MALDI experiments. CONCLUSIONS: Bionanostructure-enhanced ionization allows the analysis of a diverse selection of analytes including polymers, sugars, amino alcohols, and organic acids without interfering matrix signals. We also show that celite, a commercially available porous material containing mineralized algal bionanostructures, supports LDI-MS.

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis.

BACKGROUND AND AIMS: Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. METHODS: Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. KEY RESULTS: The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. CONCLUSIONS: The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.

Clonal aspects of plant cell proliferation and their applications to animal cells and bacteria.

OBJECTIVES: Extensive mathematical studies have been made on cell clone development but little has been advanced in the mathematics of small clone formation and virtually no actual data of small clone size has been collected. MATERIALS AND METHODS: Small clone sizes in leaf marginal cells of the aquatic plant Elodea and aleurone spot sizes in the grain of Zea were counted for later statistical analyses of mean, variance and probability distribution frequencies. RESULTS: Simple mathematical models were developed and their calculated results are comparable to data collected on actual plant clones. The parameters in these models were original cell size (s(0)), growth rate (T), duration of growth (t) and cell division inequality (i). CONCLUSIONS: Given T and t, the critical parameter is s(0). Plant tissue is ideal material to collect data on clone development because growth rate is uniform across a tissue and cells remain in place, so clone size can be measured, unlike microbes and animal cells that have neither feature. In the light of the results, traditional methods for calculating cell cycle duration and mutation rate are questioned. The applications of these plant features to studies on animal cell populations are discussed.

Cyanide-induced death of cells in plant leaves.

Destruction of guard cell nuclei in epidermis isolated from leaves of pea, maize, sunflower, and haricot bean, as well as destruction of cell nuclei in leaves of the aquatic plants waterweed and eelgrass were induced by cyanide. Destruction of nuclei was strengthened by illumination, prevented by the antioxidant alpha-tocopherol and an electron acceptor N,N,N ,N -tetramethyl-p-phenylenediamine, and removed by quinacrine. Photosynthetic O2 evolution by the leaf slices of a C3 plant (pea), or a C4 plant (maize) was inhibited by CN- inactivating ribulose-1,5-bisphosphate carboxylase, and was renewed by subsequent addition of the electron acceptor p-benzoquinone.

[Study of cytoplasm streaming as a cytophysiological method in radiolabeled experiment]. / Doslidzhennia shvydkosti rukhu tsytoplasmy iak tsytofiziolohichnyi metod v radiobiolohichnomu eksperymenti.

Estimation of influence of ionizing radiation, high-frequency electromagnetic radiation and their combined action on a higher water plant Elodea canadensis has been carried out using cytophysiological method of determination of the cytoplasm streaming rate. It was shown that low-intensive electromagnetic radiation modifies reaction of the differentiated cells on radiolesion. The rate of cytoplasm streaming can be used as an informative characteristic of plant cell state in radiobiological experiment.

Possible involvement of energy metabolism in the change of cytoplasm organization induced by a protein phosphatase inhibitor, calyculin A, in root hair cells of Limnobium stoloniferum.

In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN3, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN3. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells.

Photosynthetic and other phosphoenolpyruvate carboxylase isoforms in the single-cell, facultative C(4) system of Hydrilla verticillata.

The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C(4) plant. It typically exhibits C(3) photosynthetic characteristics, but exposure to low [CO(2)] induces a C(4) system in which the C(4) and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C(4) induction. This and enzyme kinetic data were consistent with it being the C(4) photosynthesis isoform. However, the C(4) signature serine of terrestrial plant C(4) isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C(3) sequences, was present. Western analyses of C(3) and C(4) leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C(3), non-graminaceous C(4), and Crassulacean acid metabolism PEPCs but not with the graminaceous C(4), and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C(4) angiosperms.

Femtosecond near-infrared laser pulses as a versatile non-invasive tool for intra-tissue nanoprocessing in plants without compromising viability.

In this report, we describe a highly reproducible femtosecond near-infrared (NIR) laser-based nanoprocessing technique that can be used both for non-invasive intra-tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra-precise intra-tissue nanoprocessing is achieved by the generation of high light intensity (10(12)W cm(-2)) by diffraction-limited focusing of the radiation of an NIR (lambda = 740 and 800 nm) femtosecond titanium-sapphire laser to a sub-femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non-staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non-invasively within a plant tissue. Further evidence, including two-photon imaging of chlorophyll fluorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra-fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio-temporal control of ontogenetic events and cell-cell interactions, and gravitational biology applications.